In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of fluorescent-derivatives of our tropane based DAT inhibitors. Our fluorescent tropane-based ligand, JHC1-064, has been used in many labs to characterize the trafficking and cellular distribution of SERT and DAT in living neuronal cells. Indeed, recent experiments in living neurons with JHC 1-064 have provided data that challenge mechanistic dogma for transporter translocation, as determined in DAT transfected heterologous cells, which is one area of ongoing research with these agents. This fluorescent ligand has been used in a clinical study to confirm the loss of DAT at the cell membrane in a patient with a genetically mutated DAT that was related to his Parkinsonian and attention deficit comorbidities. Recently, in order to examine the mechanisms of DAT filopodial targeting, we used quantitative live-cell fluorescence microscopy to compare the effects of the DAT inhibitor cocaine and its fluorescent analog JHC1-064 on the plasma membrane distribution of wild-type DAT and two non-functional mutants, R60A and W63A that are not accumulated in filopodia. Molecular dynamics simulations predicted that R60A preserves the outward-facing conformer to some degree through compensatory intracellular salt bridging and is therefore capable of cocaine binding. Further, JHC 1-064 binds with high affinity to the serotonin transporter (SERT) and the norepinephrine transporter (NET), as well, and we are conducting analogous studies, first in cell lines to study trafficking and cellular distribution of these other monoamine transporters. This project has led to the design of novel fluorescent ligands, using customized fluorophores suitable for live super resolution imaging. Modification of the linker between the tropane pharmacophore and the fluorophore as well as replacing the rhodamine of JHC1-064 with super bright JaneliaFluor (JF) fluorophores has resulted in novel fluorescent tools with which super resolution microscopy is currently underway.In addition, we have embarked on an SAR study with the SSRI paroxetine in order to identify high affinity and Br-containing molecules, suitable for crystallography. 4-Br-paroxetine was synthesized and compared in computational studies to paroxetine revealing the latter molecules unique binding poses that shed light on its extremely high affinity for SERT.